Project Summary: As one of the key active ingredients of tobacco smoke, nicotine exerts its actions primarily on the nicotinic acetylcholine receptors (nAChRs), which are expressed on neurons, microglia, and other cells in the central nervous system (CNS). Activation of nAChRs by nicotine has various potential therapeutic immune benefits, including modulation of inflammatory responses, maintenance of immune homeostasis, and regulation of expression of pro- and anti-inflammatory factors. However, smokers have a higher rate of HIV disease progression and behavioral and cognitive complications, with differences seen between male and female smokers. It has been suggested that prolonged exposure to nicotine in HIV patients through cigarette smoking might decrease the expression and desensitize the activation of nAChRs, which could dampen nicotine's ability to mediate immune responses. During the past several years, we have conducted a series of behavioral and molecular studies using a rodent HIV-1 transgenic (HIV-1Tg) rat model to study various medical issues, including immunity, associated with HIV patients receiving combined anti-retroviral therapy (cART). We used this HIV-transgenic rat model to determine the effects of nicotine on brain function in the presence of HIV-1 proteins and found that chronic treatment with nicotine can restore the expression of many genes altered by the HIV-1 viral proteins in signaling pathways involved in immunity and other neuronal systems. In addition, we found that expression of various nAChR subunits, especially ?6, ?3, and ?4, and immune-related genes, such as interferon regulatory factor 7 (IRF7) and interleukin-1? (IL-1?), differ greatly in HIV-1Tg rats compared with F344 controls. Our findings indicate that HIV-1 proteins greatly impact CNS immunity as well as nAChR function and alter the responsiveness to nicotine in certain immune-related and nAChR-mediated signaling pathways. Based on these findings, we hypothesize that there are significant interactions between nicotine and HIV-1 proteins that affect the immune response to nicotine in the brain of HIV-1-positive individuals. To test this hypothesis, we propose the following three aims. Aim 1 is to determine the interactive effects of nicotine and HIV viral proteins on microglia and macrophages in the brain as well as cytokine production in the blood and brain of the HIV-1Tg rat. Aim 2 is to compare the interactive effects of nicotine and HIV-1 viral proteins on gene expression in three brain regions of male and female HIV-1Tg rats using RNA-seq analysis. Aim 3 is to characterize specific genes and pathways that mediate the effects of the interaction between nicotine and HIV- 1 viral proteins using various molecular techniques, including CRISPR gene editing. The proposed studies represent the first application of next-generation sequencing technique in combination with conventional molecular approaches to investigate the modulatory effects of chronic use of nicotine on CNS immunity in the presence of HIV-1 viral proteins. By combining both genomic and molecular approaches, our studies will be comprehensive and free of subjective bias and assure the value of the data for the development of new strategies for treating HIV-1 patients who use nicotine.